Phosphorus (P) interacts with soil constituents in many ways, forming compounds of distinct stabilities in a number of chemical inorganic and organic forms. The identification of these P compounds in tropical agro-ecosystems is especially relevant to understanding potential P availability in these common P-fixing soils, providing information to improve P use efficiency. In this study, we investigated P partitioning and speciation in Brazilian Cerrado Oxisols under different management systems using a combination of sequential chemical P fractionation (SPF), P K-edge XANES and 31P NMR spectroscopy. Topsoil (0–10 cm) samples were obtained from soils under three management regimes, including native vegetation (Cerrado), no-tillage (NT), and conventional tillage (CT) agriculture. Four representative agricultural sites in the Brazilian Cerrado were selected: Ox-1 (Costa Rica-MS), Ox-2 (Sapezal-MT), Ox-3 (Cristalina-GO) and Ox-4 (Tasso Fragoso-MA). In all sites, the soils are classified as Typic Hapludox, varying in cultivation time, soil clay content and oxide types. Long-term cultivation in NT and CT led to large P accumulation in the soil. Chemical P fractionation indicated that labile P was the smallest pool at all sites. P K-edge XANES showed that P is mostly sorbed to the Fe and Al hydr(oxides), goethite, hematite and gibbsite. It also showed that phytate was the predominant organic P species, depleting in sequence from native vegetation to NT and CT management systems. 31P NMR showed that monoester P was present in all soils and management systems, whereas diester-P was found only in soils under native vegetation. The combined use of SPF, XANES and 31P NMR data was necessary to fully understand inorganic and organic P chemistry and reactivity in these tropical agricultural croplands.